oxysterol-induced DNA lysis in human leukemic cells

J. Steroid Biochem. Molec. BioL Vol. 61, No. 1/2, pp. 35-45, 1997 ~// 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain P I I : S0960-0760(96)00256-7 0960-0760/97 $17.00 + 0.00

Pergamon

Glucocorticoid/oxysterol-induced D N A Lysis in H u m a n Leukemic Cells Betty H. Johnson, Sylvette Ayala-Torres, Lee-Nien L. Chan, Mohamed E1-Naghy and E. Brad Thompson* The Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston, T X 77555-0645, U.S.A.

B o t h glucocorticoids and oxysterols, steroids with quite different k n o w n t r a n s d u c t i o n pathways, cause the death o f l y m p h o i d cells. D u a l T U N E L I p r o p i d i u m iodide assays o n sensitive h u m a n leukemic C E M - C 7 clones treated with either steroid were clearly positive by 48 h, consistent with a p o p t o sis. B o t h steroids evoked two distinctive types o f D N A lysis: cleavage into large fragments o f several different sizes and the classic " l a d d e r s " , multiples o f ~200 base pairs. C o n v e n t i o n a l gel e l e c t r o p h o r esis s h o w e d that a small p r o p o r t i o n o f total D N A had u n d e r g o n e laddering 36-48 h after t r e a t m e n t with g l u c o c o r t i c o i d or 24 h after oxysterol exposure. O n field inversion gel electrophoresis o f cellular D N A b o t h steroids c.'msed an increase in an array o f large D N A fragments <50 kb in size. A 50 kb f r a g m e n t appeared 36 h after t r e a t m e n t with either steroid, but only oxysterol t r e a t m e n t c a u s e d a significant increase in a 300 kb fragment. Oxysterol t r e a t m e n t did n o t result in D N A f r a g m e n t a t i o n in the resistant M10R5 s u b c l o n e , w h i c h retained sensitivity to glucocorticoids. We c o n c l u d e that glucocorticoids and oxysterols kill these cells with similar, but not identical, patterns o f D N A lysis w h i c h o c c u r just before or c o n c o m i t a n t with the o n s e t o f cell death. © 1997 Elsevier S c i e n c e Ltd.

J. Steroid Biochem. Molec. Biol., Vol. 61, No. 1/2, pp. 35-45, 1997

INTRODUCTION

plex set of interactions involving specific G R D N A binding sites and/or specific interactions with other transcription factors. Oxysterols do not bind to the G R or any other known m e m b e r of the steroid/thyroid/retinoid hormone receptor family. T h e precise transduction mechanism for oxysterol-induced apoptotic cell death has not been established. However, we have noted a correlation between the concentrations of oxysterols that kill C E M cells and those which occupy the oxysterol binding protein, an oxysterolspecific cytoplasmic protein which has been suggested as a receptor for oxysterol's negative regulation of cholesterol synthesis [4, 5]. Because of their different transduction pathways, each inducing cell death with the classical morphology of apoptotic cells, we have investigated the effects of oxysterols and glucocorticoids to see whether they cause D N A lysis; and if so, what fragment size/s are generated. Whereas there have been other recent studies showing D N A lysis in lymphoid cells, only one class of steroid was evaluated and only at later timepoints in the apoptotic pathway [1, 2, 6-10]. This is the first report of which we are aware comparing glucocorticoids and oxysterols in clonal h u m a n leukemic

A variety of ligands have been shown to cause changes typical of apoptosis in many cell systems. It has become clear that the steps initiating apoptotic processes differ widely from agent to agent and from cell type to cell type. However, a consensus developing in the field holds that these events may converge on one or a few final c o m m o n pathways. Among such pathways may be D N A lysis. It has been established in the C E M h u m a n leukemic lymphoid cell system that glucocorticoids and oxysterols, both derived from cholesterol, can produce apoptosis that meets the classical criteria of cell shrinkage and other morphological changes [1-3]. It has also been established that these two steroid classes initiate apoptosis by different transduction mechanisms [4]. Glucocorticoids act through and require a functional glucocorticoid receptor (GR), a ligand-activated transcription factor capable of regulating specific genes both positively and negatively through a c o m *Correspondence to E. Brad Thompson. Fax: +1 409 772 5159. Received 2 Aug. 1996; accepl:ed 25 Nov. 1996. 35

B . H . Johnson et al.

36

lymphoblasts over a wide range of time-points throughout the cell death process, following both large D N A fragmentation and traditional laddering. We have employed two clones of C E M cells: the wellstudied glucocorticoid- and oxysterol-sensitive C E M C7 clone and a glucocorticoid-sensitive, but oxysterol-resistant subclone of C E M - C 7 , M10R5. Clones of the C C R F - C E M cell line have long been recognized as a useful model system for the study of lymphocytolysis and the nature of the chemotherapeutic response in patients receiving steroid therapy [11].

MATERIALS AND METHODS

Reagents Dexamethasone (Dex) was purchased from Sigma Chemical Co. (St Louis, M O , U.S.A.) and 25hydroxycholesterol ( 2 5 O H C ) from Steraloids (Wilton, N H , U.S.A.). Both steroids were dissolved in ethanol and stored at - 2 0 ° C in the dark. R P M I 1640 was obtained from Fisher (Houston, T X , U.S.A.), fetal bovine serum (FBS) from Atlanta Biologicals (Norcross, GA, U.S.A.), trypan blue dye from Gibco/ B R L (Grand Island, NY, U.S.A.), the T U N E L assay kit from Boehringer M a n n h e i m (Indianapolis, IN, U.S.A.), and agarose from F M C (Rockland, M E , U.S.A.). T h e D N A markers were purchased from several sources: Boehringer M a n n h e i m ( D N A marker II, P F G E marker I, 2-1adder), G i b c o / B R L (Mega Base I, high molecular weight marker, 1 kb D N A ladder) and Sigma (lambda D N A single cut mixture, pulse m a r ker). Delipidated fetal bovine serum (DFBS); Dulbecco's phosphate buffered saline, p H 7.4 (PBS), and propidium iodide as well as all other chemicals were obtained from Sigma.

Cell culture T h e cells employed were derived from C C R F C E M , a h u m a n acute lymphoblastic leukemia cell line [12]. C E M - C 7 cells were isolated without selective pressure, and M 1 0 R 5 cells were obtained from C E M C7 by mutagenesis followed by selection in high concentrations of 2 5 O H C [13]. Cells were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2. All experiments were performed with cells in the logarithmic growth phase with initial cultures of 1-2 × 105 cells/ml. T h e culture m e d i u m was R P M I 1640, p H 7.4, with 5% FBS except when 2 5 O H C was used, in which case cells were transferred to R P M I 1640 with 5% D F B S to avoid the confusing effects of serum lipids; cells were maintained for 6 12 h before oxysterol addition to allow cells to adjust to the new culture conditions.

Characteristics of clones Culturing of C E M - C 7 clones over time can lead to hyperploidy and the accumulation of spontaneous

steroid-resistant mutants. T o avoid these problems both C E M - C 7 and M 1 0 R 5 cells were cloned in soft agar during the early stages of this study. A fresh Dex-sensitive, diploid clone of C E M - C 7 was chosen. Its ploidy was determined by Giemsa staining of mitotic cells' chromosomes, and all 75 cells evaluated were diploid. M 1 0 R 5 was selected from C E M - C 7 for its resistance to 2 5 O H C , and this clone retained Dex sensitivity. M 1 0 R 5 cells were ~ 7 6 % hyperploid (18 cells out of 75 were diploid). T h e doubling time of C E M - C 7 cells in m e d i u m containing 5% FBS was 23.1 + 1.5 h and 20.8 + 0.5 h in 5% DFBS. M 1 0 R 5 cells exhibited similar doubling times: 26.5 + 1.8 h in m e d i u m with 5% FBS and 22.7 + 0.9 h in m e d i u m with 5% D F B S ( n - - 4 - 6 determinations for each clone). T h e m e a n diameter of control cells was evaluated on a Coulter 256 Channelyzer (Coulter Source, Marietta, GA, U.S.A.). C E M - C 7 cells had a m e a n diameter of 11.2 + 2.8 # m in 5% FBS and 11.6 ± 2.4 p m in 5% D F B S , whereas M 1 0 R 5 cells in 5% FBS had a m e a n diameter of 12.8 + 5.1 #m, and in 5% D F B S 14.0 + 4.4/~m. T h e n u m b e r of cells evaluated in the cell size studies ranged from 5700 to 8275.

Analysis of apoptosis by flow cytometry The terminal deoxynucleotidyl transferasemediated d U T P - X nick end labeling ( T U N E L ) assay was performed according to the protocol provided by the manufacturer (Boehringer M a n n h e i m ) with some modifications. Before fixation, 3 x 106 cells were centrifuged at 1 0 0 0 r p m for 1 0 m i n and washed twice with PBS containing 1% bovine serum albumin. T h e cells were fixed using 4% freshly prepared paraformaldehyde in PBS and incubating at 22°C for 3 0 60 min. T h e fixed cells were washed, permeabilized, washed again and labelled with a mixture containing the terminal deoxynucleotidyl transferase and modified d U T P for 60 min at 37°C. Each assay contained two negative controls which did not receive the transferase enzyme and two positive controls in which D N a s e I was added for a 10 min 22°C incubation. After washing, all samples were stained with propidium iodide according to a m e t h o d similar to that described by Van H o u t e n et al. [14]. Cells were initially treated with a low salt staining solution (3% polyethylene glycol 8000, 50 ~g/ml propidium iodide, 180 units/ml RNase A, 0.1% T r i t o n - X 100 in PBS/ 1% sodium azide, 4 m M sodium citrate) and incubated at 37°C in the dark for 20 min with gentle mixing. After adding a high salt staining solution (3% polyethylene glycol 8000, 50 #g/ml propidium iodide, 0.1% T r i t o n - X 100, 4 0 0 m M sodium chloride) and mixing, cells were incubated overnight in the dark before analysing by flow cytometry. Samples (20 000 cells) were analysed for D N A content and D N A breaks by flow cytometry using a F A C S c a n (Becton Dickinson, Bedford, MA, U.S.A.). F o r the T U N E L

Glucocorticoid/oxysterol-induced DNA Lysis analysis, the Cell Quest 1.2 (Becton Dickinson) software was used. Analysis was done using an argon-ion laser with excitation at 488 nm. Doublets and cell aggregates were gated out and only the singlet cell population was analysed. Red (propidium iodide) and green (fluorescein-dUTP) fluorescences were detected using 530 n m and 584 n m filters, respectively. Flow cytometry was initially conducted with propidium iodide staining only.

Analysis of DNA nucleosomal units by conventional gel electrophoresis Cells were cultured in the appropriate media and treated with ± 1 / ~ M D e x or ± 1 / ~ M 2 5 O H C for 48 h. T h e protocol for isolating and separating small D N A fragments by gel electrophoresis was a modification of D u k e and C o h e n ' s m e t h o d [15]. Cell pellets from 2 x 106 cells were washed once with PBS, treated with NP-40, vortex mixed, centrifuged; then 5 M NaC1 and isopropanol were added to the cell-free supernatant. All procedures were performed at 4°C. T h e D N A precipitate was washed with 70% ethanol, dried, and resuspended in T E buffer, p H 7.4 (10 m M T r i s - H C 1 , 1 m M ethyllene diamine tetraacetic acid ( E D T A ) ) . After adding the loading buffer, samples were heated to 65°C for 10rain, and applied to a 1.2% agarose gel. A G i b c o / B R L 1 kb D N A ladder was loaded to the center lane of the gel to size the D N A fragments. Electrophoresis was conducted at 50 V for 2 h at 22°C in a submarine gel unit with a running buffer of 1 × T B E ( 0 . 0 9 M Tris-borate, 2 m M E D T A ) . Ethidium bromide (0.5 #g/ml) was used to stain the D N A , which was visualized at 3 0 2 n m U V and photographed with type 667 Polaroid film.

Analysis of large DNA fragments by field inversion gel electrophoresis Cells were cultured in the appropriate media and treated with +1 p M Dex or ___1# M 2 5 O H C for varying periods of time. F o r analysis of large D N A fragments, a modified m e t h o d of A m a n d and Southern [16] was followed. Cells (4 x 106) were washed once in 22°C PBS, e m b e d d e d in 0.6% agarose at 45°C, cooled to 4°C in a 10-well plug mold from BioRad (Hercules, CA, U.S.A.). T h e plugs containing the cell gels were transferred to a lysis buffer (1% sarkosyl, 50 m M E D T A , 50 m M T r i s - H C l ) containing 0.2 mg/ ml proteinase K and incubated overnight at 45°C. T h e plugs were rinsed three times with T E buffer, and stored at 4°C in T E . For D N A analysis, the plugs were inserted into wells in a 1% agarose gel prepared with 0.5 x T B E running buffer, sealed in place with 1% agarose, and electrophoresed on a BioRad horizontal Sub Cell gel unit (15 x 15 cm), using a BioRad 200/2.0 power ,supply, a Wiz p u m p (Isco, Lincoln, NE, U.S.A.) to recirculate buffer between the chambers, and an M. J. Research p r o g r a m m a b l e

37

power inverter (Cambridge, MA, U.S.A.). For D N A fragments <50 kb, program 2 ( m i n i m u m reverse time 0.01 s, m a x i m u m 0.59 s, 3068 ramps) was used at 7.5 V/cm for 1 4 h 49 min at 14°C, for D N A fragments from 50 to 3 0 0 k b , p r o g r a m 5 ( m i n i m u m reverse time 0.1 sec, m a x i m u m 10 s, 116 ramps) at 6 V/cm for 20 h 14 min at 10°C; and for D N A fragments >300 kb, program 6 ( m i n i m u m reverse time 0.1 s m a x i m u m 10 s, 39 ramps) at 6 V / c m for 22 h 8 min at 23°C. T h e various D N A size markers used are listed under Reagents. Gels were stained with ethidium bromide (0.5 #g/ml) for 2 - 3 h, destained in water for 1 h, visualized at 254 n m U V light and photographed with type 55 Polaroid film. Image analysis for quantitation of D N A fragments was carried out on the negatives of the Polaroid film by the Lynx 5000 densitometer on the Applied Imager system.

RESULTS

Growth responses of CEM-C7 and M1 OR5 cells to glucocorticoid vs oxysterol T o demonstrate the biological responses of the freshly isolated C E M - C 7 and M 1 0 R 5 clones to the two classes of steroids, cells in logarithmic growth were exposed to a lethal concentration of each steroid and their growth and viability were followed for 72 h (Fig. 1). Both C E M - C 7 and M 1 0 R 5 cells were killed by glucocorticoids. In cultures treated with 1 ~tM Dex, cell viability remained high for 24 h, and extensive cell death was observed >48 h after the addition of the drug, so that by 72 h the n u m b e r of surviving cells in the treated culture was reduced by almost half from the original seeded level, and represented only 8% c o m p a r e d to the control, logarithmically growing culture. Longer incubation led to virtually complete death of the cells (data not show). With oxysterol treatment, a similar but more rapidly lethal pattern in C E M - C 7 cells was observed. As with Dex-treated cells, there was a delay of ~ 2 4 h after the addition of 2 5 O H C during which the cells grew as well as controis. After that, increasing cell death was seen, so that by 72 h the original cell numbers had been reduced by two orders of magnitude. T h e M 1 0 R 5 clone was completely resistant to the oxysterol. At the 48 h time-point, cell size was determined by Coulter Channelyzer. M e a n cell diameter was significantly reduced in cells sensitive to 1 ~ M Dex or 2 5 O H C , whereas no significant change in m e a n cell diameter was observed in the 25OHC-resistant M 1 0 R 5 cells (Table 1). Because these cells are pres u m e d to be essentially spherical in shape, one m a y conclude that a reduction in m e a n cell diameter indicates a decrease in cell volume. Cell shrinkage has clearly been established as a morphological characteristic of apoptosis [17], and this criterion was m e t by

38

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C E M - C 7 as well as M 1 0 R 5 cells at this concentration of either steroid. Apoptotic cell responses o f C E M - C 7 and M 1 OR5 cells to glucocorticoid vs oxysterol

Flow cytometric analysis of propidium iodidestained cells has been used to measure apoptosis directly. Apoptotic nuclei appear as a peak of hypodiploid D N A when cells are stained with DNA-specific Table 1. Effect on cell diameter after 48 h 1 # M drug treatment

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fluorochromes [18]. C E M - C 7 and M 1 0 R 5 cells cultured in the appropriate media were treated with 1 # M Dex or 2 5 O H C for 48 h, stained with propidium iodide and evaluated by flow cytometry. Figure 2, panels A and B, represent the results from the two cell clones treated with 1 p M Dex. T h e profiles of D N A content show the usual three phases of the cell cycle (G0/G1, S, G 2 / M ) in the control cells. T h e right hand panels show a clear increase in the hypodiploid population in both glucocorticoid-treated clones. C E M - C 7 cells present the profile of a typical diploid cell whereas M 1 0 R 5 cells are mostly hyperploid. T h e Dex-treated cells also have more cells in the G0/G1 cell cycle phase than do control cells (49% vs 47% for control C E M - C 7 cells and 61% vs 51% for control M 1 0 R 5 cells), consistent with our earlier report [19]. Panels C and D show the results of exposure of the same clones to 2 5 O H C for 48 h. In C E M - C 7 cells the fraction of cells in the G0/G1 phase was drastically reduced (47% for control C E M C7 cells to 3%), whereas it is unaltered in the insensitive M 1 0 R 5 cells (54% for control M 1 0 R 5 cells to 56%). In unpublished preliminary experiments, we found that shorter treatment with 2 5 O H C caused the accumulation of C E M - C 7 cells in the G0/G1 phase. It is likely that oxysterol treatment in this experiment did initially cause an increase in the n u m b e r of cells in G0/G1, but at the time evaluated here (42h), those cells had been eliminated. T h e hypodiploid, apoptotic cells were increased significantly only in the oxysterol-sensitive C E M - C 7 clone. A flow cytometric T U N E L assay combined with a secondary stain for total D N A was used to detect both single-stranded nicks and double-stranded breaks in D N A , together with cell cycle distributions. In Fig. 3 cell cycle distributions are shown as contours, with G0/G1, S and G 2 / M in the same relative positions on the abscissa as in Fig. 2. T h e negative controls averaged 0.3 ± 0.2% apoptotic cells, whereas the positive controls averaged 86.4_+6.8%, n = 5 (data not shown). In panels A - D of Fig. 3, the data on the left show the control C E M - C 7 and M 1 0 R 5 cells. These graphs demonstrate that for both C E M C7 and M 1 0 R 5 clones transferring the cells from FBS to D F B S did not alter the contour profiles. Both Dex-treated C E M - C 7 cells (panel A) and M 1 0 R 5 cells (panel B) show T U N E L - p o s i t i v e cells as areas which appear above the normal contour, and these represent 7% and 10.9% apoptotic cells, respectively. In contrast, oxysterol-treated C E M - C 7 cells show extensive D N A damage (panel C with 51.1% apoptotic cells), whereas M 1 0 R 5 cells show essentially none (panel D). By this test, D N A damage consistent with apoptotic cell death, is thus seen after exposure of sensitive cells to either steroid.

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l ig. 2. A p o p t o t i c ceils i n c r e a s e d in b o t h C E M - C 7 a n d M10R5 cells e x p o s e d to g l u c o c o r t l c o i d , a n d only in ( E M - C 7 cells e x p o s e d to oxysterol. All cells w e r e c u l t u r e d a n d t r e a t e d as in Fig. 1. A f t e r 42 h cells w e r e - l a i n e d w i t h p r o p i d l u m iodide a n d e v a l u a t e d b y flow c y t o m e t r y . Cell cycle D N A h i s t o g r a m s o f c o n t r o l a n d ~realed cells a r e ~;hown on t h e left a n d r i g h t o f e a c h p a n e l , r e s p e c t i v e l y . P a n e l A = C E M - C 7 a n d P a n e l It = XI10R5 t r e a t e d w i t h 1/tM D e x ; P a n e l C = C E M - C 7 a n d P a n e l D = M10R cells t r e a t e d w i t h 1 #M 2 5 O H C . T h e pe:rcentage o f a p o p t o t i c cells is given on t h e g r a p h i c s to t h e left o f t h e G0/G1 p e a k s .

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Fig. 3. D N A n i c k i n g o c c u r r e d i n b o t h C E M - C 7 a n d M 1 0 R 5 ceils e x p o s e d to g l u c o c o r t i c o i d , a n d o n l y i n C E M C7 ceils e x p o s e d to o x y s t e r o l . All ceils w e r e c u l t u r e d a n d t r e a t e d a s in Fig. 1. A f t e r 48 h cells w e r e l a b e l e d a t DNA strand breaks with fluorescein dUTP using the terminal deoxynucleotidyl transferase, and the total DNA w a s s t a i n e d w i t h p r o p i d i u m i o d i d e . A f t e r flow c y t o m e t r y , c o n t o u r g r a p h s o f c o n t r o l a n d t r e a t e d ceils a r e s h o w n i n t h e left a n d r i g h t s i d e s o f e a c h p a n e l , r e s p e c t i v e l y . P a n e l A = C E M - C 7 a n d P a n e l B = M 1 0 R 5 ceils +_1 p M D e x ; P a n e l C = C E M - C 7 a n d P a n e l D = M 1 0 R 5 ceils + 1 p M 2 5 O H C . T h e t o t a l p e r c e n t a g e o f a p o p t o t i c ceils is i n d i c a t e d o n t h e g r a p h s .

G l u c o c o r t i c o i d / o x y s t e r o l - i n d u c e d D N A Lysis

D N A fragmentation into "'DNA ladders" occurred to a limited extent in C E M cells sensitive to glucocorticoid or oxysterol D N A f r a g m e n t a t i o n has t r a d i t i o n a l l y b e e n e v a l u a t e d b y gel e l e c t r o p h o r e s i s , w h e r e t h e d i s p l a y o f D N A fragm e n t s into " l a d d e r s " r e p r e s e n t i n g m u l t i p l e s of-,~200 b p

A +

41

h a s s o m e t i m e s b e e n t a k e n as a definitive m a r k o f a p o p tosis. W e s h o w h e r e t h a t C E M - C 7 a n d M 1 0 R 5 cells t r e a t e d w i t h D e x for 48 h d o lyse a small a m o u n t o f D N A t h a t c a n b e seen as ~ 2 0 0 b p l a d d e r s , w i t h a s m e a r of larger-sized DNA. Only a small amount of such D N A d a m a g e is s e e n at 36 h in C E M - C 7 cells (Fig. 4,

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Fig. 4. L a d d e r s o f s m a l l D N A f r a g m e n t s w e r e e v i d e n t in g l u c o c o r t l c o i d - t r e a t e d C E M - C 7 a n d MIOR5 cells, a n d also i n o x y s t e r o l - t r e a t e d C E M - C 7 cells. All cells w e r e c u l t u r e d a n d t r e a t e d as in Fig. 1. P a n e l A s h o w s t h e D N A p a t t e r n f o r C E M - C 7 cells w i t h (+) a n d w i t h o u t (-) 1 p M D e x t r e a t m e n t o v e r t h e t i m e c o u r s e o f 48, 36, 24, a n d 12 h. P a n e l B is t h e s a m e e v a l u a t i o n f o r M10R5 cells, 1 p M D e x . T h e r e s u l t s f r o m o x y s t e r o l t r e a t m e n t o f C E M - C 7 cells is s h o w n in P a n e l C a n d MIOR5 cells in P a n e l D. T h e c e n t e r l a n e in all gels s h o w s D N A size m a r k e r s w i t h t h e a p p r o p r i a t e size given f o r P a n e l A.

42

B.H.

J o h n s o n et al.

panel A), but faint laddering of D N A from M 1 0 R 5 cells is seen at 3 6 h (panel B). C E M - C 7 cells showed a similar D N A ladder pattern after 24 h treatment with 1 p M 2 5 O H C (panel C), which intensified over time. The oxysterol-resistant M 1 0 R 5 cells revealed no evidence of such D N A damage (panel D); both C E M - C 7 and M 1 0 R 5 cells cultured in D F B S showed a faint smear of D N A in control cultures at all time-points. In FBS cultures this was seen to a m u c h lesser extent.

A

D N A fragmentation into large discrete sizes occurred in cells sensitive to glucocorticoid and oxysterol Conventional agarose gel electrophoresis can be used to detect D N A fragments up to ~ 5 0 kb in size, but by field inversion electrophoresis D N A fragments as large as 2 0 0 0 k b can be identified [16]. We have analysed C E M cell D N A after treatment with Dex or 2 5 O H C , varying the conditions of electrophoresis so as to evaluate D N A sizes from 10 to 1000 kb. Either glucocorticoid and oxysterol caused D N A breakage into discrete large fragments. Figure 5, panels A - D ,

B 18

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Fig. 5. L a r g e D N A f r a g m e n t a t i o n o c c u r r e d i n g l u c o c o r t i c o i d - t r e a t e d C E M - C 7 a n d M 1 0 R 5 cells a n d i n o x y s t e r o l - t r e a t e d C E M - C 7 , a n d n o t M 1 0 R 5 cells. All cells w e r e c u l t u r e d a n d t r e a t e d a s in Fig. 1. T h e r u n c o n d i t i o n s u s e d h e r e e v a l u a t e d D N A f r a g m e n t s i n t h e 10 to 100 k b r a n g e . P a n e l A s h o w s t h e D N A p a t t e r n f o r C E M - C 7 cells, P a n e l B f o r M 1 0 R 5 c e l l s t r e a t e d f o r 18, 24, 36, a n d 48 h w i t h 1 / t M D e x ( c o n t r o l l a n e f i r s t foll o w e d b y t h e t r e a t e d l a n e ) . P a n e l C s h o w s t h e D N A p a t t e r n f o r C E M - C 7 cells, P a n e l D f o r M 1 0 R 5 c e l l s t r e a t e d f o r 18, 24, 36, 48 h w i t h 1 p M 2 5 O H C .

Glucocorticoid/oxysterol-induced DNA Lysis shows the results from one experiment in which conditions were selected to 'visualize D N A bands from 10 to 100 kb. T h e patterns differed slightly in detail for the two types of steroids. All samples, both control and treated, contained two discrete D N A bands, one ~ 5 0 kb and one slightly larger. In the samples of glucocorticoid-treated sensitive cells, only the lower b a n d intensified over time. In oxysterol-treated sensitive cells, after 24 h both bands were increased over time when c o m p a r e d to controls (5C). In addition after either steroid was added, a D N A smear from ~8 to 35 kb, with a m a x i m u m intensity at ~15 kb was consistently seen at the times sampled after >24 h treatm e n t (5A-C). Efforts to resolve distinct bands of D N A < 5 0 k b were unsuccessful. Only faint D N A bands were observed in the oxysterol-insensitive M 1 0 R 5 cells treated with 2 5 O H C (5D). W h e n conditions of analysis were :adjusted to display D N A fragments in the 100-1000 kb size range, a faint b a n d at ~300 kb was apparent in D N A from cells under all conditions. After steroid treatment, this band increased only in the sensitive C E M - C 7 cells exposed to 2 5 O H C for >24 h (data not shown). N o significant D N A bands were detected above 300 kb. W h e n the photographic data from two to four experiments for each condition were converted to numerical data by image: analysis and averaged, the temporal patterns of these changes in D N A fragments could be seen m o r e clearly. In Fig. 6, panels A and B show the results of De,x treatment of the two glucocorticoid-sensitive clones, and panel C shows the results of 2 5 O H C treatment of the oxysterol-sensitive clone. In all sensitive cells with either steroid treatment, the greatest increase in D N A fragmentation occurred in the smear of D N A of the <50 kb sizes which clearly b e c a m e apparent after 24 h. In clone M 1 0 R 5 (panel B), the <50 kb D N A smear seems to appear somewhat earlier than in C E M - C 7 , beginning to diverge from controls at or before 24 h in Dex. Neither glucocorticoid-.sensitive clone showed any change in the 300 kb bands, and the 50 kb b a n d only manifested itself when significant numbers of apoptotic cells b e c a m e apparent, i.e. after _>36 h in Dex. T h e pattern of D N A lysis in C E M - C 7 cells after 2 5 O H C treatment was somewhat different. Although in the experiment shown in Fig. 5 (panel C) the <50 kb b a n d appears greater than controls as early as at 18 h, these results were not consistent so that on average we found no change at that time. T h e 50 kb and 300 kb bands suggest a two-fold increase over controls at 18 h, followed by a return to control levels at 2 4 h , but the range of variation around the 18 h time-point m a d e any firm conclusion impossible. Thereafter, all three sizes of D N A increased progressively, reflecting the increasing numbers of dead cells.

43

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HOURS AFTER TREATMENT Fig. 6. T h e i n c r e a s e in D N A f r a g m e n t s o f 8 to 35 kb a n d t h e 50 kb b a n d in cells s e n s i t i v e to g i u c o e o r t i c o i d a n d o x y s t e r o l t r e a t m e n t a n d t h e 3 0 0 k b b a n d in t h e o x y s t e r o l - s e n s i t i v e cells. D e n s i t o m e t r i c s c a n n i n g o f t h e n e g a t i v e s of t h e p h o t o g r a p h s in Fig. 5 as well as p h o t o g r a p h s o f o t h e r a s s a y s w e r e p e r f o r m e d . In e a c h a s s a y t h e c o n t r o l D N A b a n d s for a p a r t i c u l a r s i z e w e r e a v e r a g e d a n d t h e f o l d - l n c r e a s e o f t h e treate d b a n d w a s d e t e r m i n e d b y d i v i d i n g that b a n d ' s i n t e n s i t y b y the average control band intensity. Panel A represents a time c o u r s e o f t h r e e s i z e s o f D N A f r a g m e n t s o f C E M - C 7 cells t r e a t e d w i t h 1/~M D e x a n d P a n e l B, M 1 0 R 5 cells. In P a n e l C t h e D N A f r a g m e n t a t i o n p a t t e r n o f C E M - C 7 cells t r e a t e d w i t h 1~ 2 5 O H C is given. E r r o r b a r s r e p r e s e n t t h e s t a n d a r d d e v i a t i o n , n = 2-3.

DISCUSSION

T h e primary purpose of this study was to determine whether, when, and what sizes of D N A fragmentation occurred during the apoptotic death of C E M cells caused by two differing classes of steroids: glucocorticoids and oxysterols. By doing so we wished to test the specificity of action of each steroid, and to determine whether they evoked the postulated "universal final pathway". O u r earlier studies and those of others indicated that even though these two types of steroids initiated cell death by different transduction mechanisms, the cells followed a similar kinetic pattern to their death. This included cell shrinkage beginning relatively early after exposure to either steroid (measurable by Coulter Channelizer after ~ 6 h ) , cell

44

B . H . Johnson

growth continuing for ~ 2 4 h, trapping of cells in GO/ G1 phase of the cell cycle, and increasing cell death becoming apparent at the times of continued incubation in steroid beyond 24 h. During this 24 h delay period, removal of the agonist steroid, or addition of antagonists reversed cell shrinkage and completely prevented cell death [19, 20]. T h r e e types of evidence are invoked here to support the conclusion that apoptosis is the type of cell death caused by both steroids: cell diameter, flow cytometric analyses of propidium iodide stained cells, and D N A lysis. O f course it is well known that glucocorticoids cause the apoptotic death of lymphoid cells in general, and in sensitive clones of C E M cells in particular. Our results are consistent with such cell death from glucocorticoids in the case of these recently cloned C E M cells. Only a few reports have suggested that oxysterols cause apoptotic cell death [2, 6], and the data here extend those reports. Both the T U N E L assay and the propidium iodide flow cytometry assay depend to some degree on D N A lysis, and therefore of themselves cannot completely prove that the cell death observed is apoptotic. T h e primary definition of apoptosis is morphological, and to our knowledge no complete account of the morphological changes in lymphoid cells caused by oxysterols has yet been p u b lished. We have carried out such an analysis for C E M - C 7 cells, and in data to be published separately found that 2 5 O H C indeed causes classic apoptotic changes [3]. We can thus conclude that the D N A lytic changes which we see here are associated with an apoptotic process. Earlier work had shown that D N A lysis into fragm e n t s whose sizes are multiples of those protected by nucleosomes (~200 bp) occurred, to a limited extent and late in the process, after glucocorticoid treatment of C E M - C 7 cells [1, 7, 8]. Our results confirm and extend those findings and show that these D N A "ladders" are demonstrable in this subclone of C E M - C 7 cells after 48 h in Dex, at a time when significant numbers of the cells are dead or dying. T h e M 1 0 R 5 clone shows D N A laddering by 3 6 h in Dex, suggesting that these cells are slightly m o r e sensitive to Dex treatment. T h e C E M - C 7 cells in 2 5 O H C showed D N A ladders faintly at 24 h and clearly by 3 6 h after addition of the steroid, indicating that 2 5 O H C may be a m o r e potent inducer of apoptosis than Dex in C E M - C 7 cells, or that the D N A lysis portion of the apoptotic pathway m a y differ between the two steroids. Although initial research on apoptotic-related D N A lysis concentrated on internucleosomal fragmentation, in the early 1990s reports appeared showing that in rat thymocytes glucocorticoids rapidly caused D N A lysis into large, discrete fragments of ~ 5 0 and 300 kb [21]. It was noted that these sizes were quite similar to those of D N A involved in specific chromatin structures, namely loops of 50 kb and rosettes of 300 kb

et al.

[22]. It has been shown that in glucocorticoid-evoked apoptosis in rat thymocytes, when Zn ÷2 was added to the m e d i u m , D N A was cleaved into large fragments only, without internucleosomal cleavage [21]; whereas in the h u m a n leukemic cell line, U937, co-treatment with zinc inhibited D N A fragmentation and prevented apoptosis [9]. T h e proposal has been m a d e that D N A cleavage into large pieces precedes cell death and lysis of D N A into nucleosome-sized ladders [10, 23-26]. T h e data from two clones of C E M - C 7 cells treated with Dex failed to reveal the development of a 300 kb fragment as reported earlier [1]. Fragments of ,~50 kb accumulated following Dex treatment for > 3 6 h , when cells were clearly dying. A dramatic accumulation of a broad range of D N A fragments seen as a smear of unresolved bands with the greatest intensity around 15 kb was also seen. This appears to diverge from controls between 24 and 36 h after addition of the steroid. Therefore the sequence and some fragments seen in apoptotic rat thymocytes are not exactly replicated in this h u m a n leukemic lymphoid cell system. With oxysterol treatment, the sensitive C E M - C 7 cells also show D N A lysis into various sizes. T h e T U N E L assay indicates that the oxysterol induces m u c h m o r e extensive D N A damage than that caused by the glucocorticoid. In addition, field inversion gel electrophoresis data show an increase in a 300 kb fragment as well as the ~50 kb fragment and the <50 kb D N A smear, all of which are clearly and reproducibly seen after 36 h treatment. As with glucocorticoids, the appearance of these larger fragments is not definitively earlier than that of internucleosomal ladders. T h e s e manifestations of nucleolysis seem to b e c o m e clear only as large numbers of cells are dying, although interpolation of the curves between data points suggests that the processes m a y begin before cell death. We conclude that lysis of D N A into several more or less discrete size ranges, both large and small, occurs in both the glucocorticoid- and oxysterolevoked apoptotic cell death of sensitive clones of C E M cells. In the clones studied here, selection for resistance to oxysterol was accompanied by loss of the nucleolytic responses. Our data are as consistent with simultaneous lysis of D N A into both large and nucleosome-sized fragments as it is with a precursorproduct relationship between the large and small pieces. It is also clear that D N A lysis into certain relatively discrete fragments is an end-stage result of the apoptotic death pathway brought on by these two classes of steroids acting through entirely different initial transduction mechanisms, and that D N A lysis into similarly sized fragrnent/s is a c o m m o n portion of that pathway. Although there is still debate in the literature concerning the nature of the apoptotic pathway caused by steroids, our work supports the hypotheses that: (a) h u m a n malignant lymphoid cells

Glucocorticoid/oxysterol-induced DNA Lysis do not follow the identi~-al death mechanisrn/s as normal thymocytes, and (b) D N A fragmentation in malignant lymphoid cells occurs rather late in the pathway and not necessarily by a progression of large size fragments to smaller ones. Acknowledgements--We wish to acknowledge the assistance of Drs William Plunkett and Peng Hlmg at The University of Texas M. D. Anderson Cancer Center, Houston, TX, and Dr Michael Finney of MJ Research, Cambridge, M~, for their assistance in developing the protocols for field inversion ~:el electrophoresis evaluation of large DNA fragments. We also wish to recognize Dr Feng Zhou and Mark Griffin, The University of Texas Medical Branch, Galveston, TX for their assistance in preparing graphs and figures. This work was supported by N I H - N I D D K grant 5P01-DK 42788.

REFERENCES 1. Wood A. C., Waters C . M . , Garner A. and Hickman J. A., Changes in c-myc expre:~sion and the kinetics of dexamethasone-induced programmed cell d e a t h (apoptosis) in human lymphoid leukaemia cell,;. Br. J. Cancer 69 (4) (1994) 663669. 2. Christ M., Luu B., Mejia J. E., Moosbrugger I. and Bischoff P., Apoptosis induced by oxysterols in murine lymphoma cells and in normal thymocyte!~. Immunology 78 (1993) 455-460. 3. Ayala-Torres S., Moiler P., Johnson B. H. and Thompson E. B.: Characteristics of 25.-hydroxycholesterol-induced apoptosis in a human leukemic cell line. Exp. Cell. Res. (in press). 4. Bakos J. T., Johnson B. H. and Thompson E. B., Oxysterolinduced cell death in human leukemic T-cells correlates with oxysterol binding protein occupancy and is independent of gincocorticoid-induced apol:,tosis. J. Steroid Biochem. Molec. BioL 46 (1993) 415-426. 5. Taylor F. R., Correlation among oxysterol potencies in the regulation of the degradation of 3-hydroxy-3-methylglutaryl CoA reductase, the repression of 3-hydroxy-3-methylglutaryl CoA synthase and affinities for the oxysterol receptor. Biochern. Biophys. Res. Commun. 1~16 (1992) 182-189. 6. Aupeix K., Weltin D., Mejia J. E., Christ M., Marchal J., Freyssinet J.M. and Bischoff P., Oxysterol-induced apoptosis in human monocytic cell lines. Immunobiology 194 (1995) 415428. 7. Alnemri E. S. and Litwack G., Glucocorticoid-induced lymphocytolysis is not mediated by an induced endonuclease. ft. Biol. Chem. 264 (1989)4104-4111. 8. Alnemri E. S. and Litwack G., Activation of intemucleosomal DNA cleavage in human CEM lymphocytes by glucocorticoid and novobiocin, ft. Biol. Chem. 265 (1990) 17323-17333. 9. Bicknell G. R., Snowden R. T. and Cohen G. M., Formation of high molecular mass I)NA fragments is a marker of apoptosis in the human leukaemic cell line, U937. J. Cell Sci. 107 (1994) 2483-2489. 10. Cohen G . M . , Sun S. lvl., Fearnhead H., MacFarlane M., Brown D. G., Snowden R. T. and Dinsdale D., Formation of

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45

large molecular weight fragments of DNA is a key committed step of apoptosis in thymocytes, ft. lmrnunol. 153 (1994) 507516. Harmon J. M., Norman M. R. and Thompson E. B.~ Human leukemic cells in culture - - a model system for the study of glucocorticoid-induced lymphocytolysis. In Steroid Receptors and the Management of Cancer (Edited by E. B. Thompson and M. E. Lippman). CRC Press, Boca Raton, FL (1979) pp. 113129. Norman M. R. and Thompson E. B., Characterization of a glucocorticoid-sensitive human lymphoid cell line. Cancer Res. 37 (1977) 3785-3791. Ayala-Torres S., Johnson B. H. and Thompson E. B., Oxysterol sensitive and resistant lymphoid cells: correlation with regulation of cellular nucleic acid binding protein mRNA. ft. Steroid Biochem. Molec. Biol. 48 (1994) 307-315. Van Houten N. and Budd R. C., Accelerated programmed cell death of MRL-lpr/lpr T lymphocytes. J. Immunol. 149 (1992) 2513-2517. Duke, R. C. and Cohen, J. J., Morphological and biochemical assays of apoptosis. In Current Protocols in Immunology, eds J~ E. Coligan, A. M. Kruisbeek, D. H Margulies, E. M. Shevach and W. Strober. Greene Publishing and Wiley-Interscience, New York, 1992, pp. 3.17.1-3.17.16. Anand, R. and Southern, E . M . , Pulsed field gel electrophoresis. In Gel Electrophoresis of Nucleic Acids: a Practical Approach, 2nd e d n , eds D. Rickwood and B. D. Hames. IRL Press, Oxford, U.K., 1990, pp. 101-123. Wyllie A. H., Apoptosis: cell death in tissue regulation. ft. Pathol. 153 (1987) 313-316. Nicoletti I., Migliorati G., Pagliacci M. C., Griguani F. and Riccardi C., A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Irnrnun. Methods. 139 (1991) 271-279. Harmon J. M., Norman M. R., Fowlkes B. J. and Thompson E. B., Dexamethasone induces irreversible G1 arrest and death of a human lymphoid cell line. ft. Cell. Physiol. 98 (1979) 267278. Benson R. S. P., Heer S., Dive C. and Watson A. J.M., Characterization of cell volume loss in CEM-C7A cells during dexamethasone-induced apoptosis. Am. ft. Physiol. 270 (1996) C1190-C1203. Brown D. G., Sun X.-M. and Cohen G. M., Dexamethasoneinduced apoptosis involves cleavage of DNA to large fragments prior to intemucleosomal fragmentation. J. BioL Chem. 268 (1993) 3037-3039. Filipski J., Leblanc J., Youdale T., Sikorska M. and Walker P. R., Periodicity of DNA folding in higher order chromatin structures. E M B O J . 9 (1990) 1319-1327. Squier M. K. T., Sehnert A. J. and Cohen J. J., Apoptosis in leukocytes, ft. Leukoc. Biol. 57 (1995) 2-10. Eamshaw W. C., Nuclear changes in apoptosis. Curr. Opin. Cell Biol. 7 (1995) 337-343. Evans-Storms R. B. and Cidlowski J. A., Regulation of apoptosis by steroid hormones. J. Steroid Biochern. Molec. BioL 53 (1995) 1-8. Ioannou Y. A. and Chen F. W., Quantitation of DNA fragmentation in apoptosis. Nucl. Acids Res. 24 (1996) 992-993.